Development and differentiation require that individual cells make appropriate cell fate decisions in a coordinated fashion. Intercellular signaling is an integral part of this coordination. A small number of extracellular ligands play key roles in this process. The proteins encoded by the Drosophila gene wingless (wg) and its vertebrate ortholog Wnt-1 are two such signals. The wg/Wnt-1 pathway regulates both differentiation and cell proliferation. wg/Wnt-1 signaling modulates the activity of several downstream target proteins, including APC, a tumor suppressor gene implicated in the onset of many colon cancers. Misregulation of the genes in this pathway can also lead to the onset of cancer. For example ectopic expression of Wnt-1 in mice has been shown to induce breast carcinomas. The long term goals of this work are to understand the steps leading to post-translational modification and secretion of the ligand; the factors that govern ligand-receptor recognition and activation; and the role extracellular glycosaminoglycans play in signaling. A variety of biochemical and molecular-genetic tools will be used to examine the interactions between wg protein (WG), extracellular proteoglycans, and potential receptors. The affinity of WG binding to Drosophila cells expressing either DFRIZZLED2 or DFRIZZLED1 will be measured using single cell fluorescence binding assays; immunoprecipitation and affinity chromatography techniques will be used to test for direct WG-FRIZZLED binding; and the effects of extracellular glycosaminoglycans on receptor binding, WG activity, and extracellular localization will be tested.